Extended type 1 chain glycosphingolipids as tumor-associated antigens

ABSTRACT

A variety of compounds are provided which are usefdl as immunogens and as tumor markers. The present invention discloses methods relating to the detection of cancer. Extended forms of the lacto-series type 1 chain are shown to be present in various cancer tissues. The present invention also provides a cell line and the monoclonal antibody produced therefrom. Such an antibody has a number of uses, including in diagnostic or therapeutic methods.

CROSS REFERENCE TO RELATED APPLICATION

This is a Rule 62 Continuation-in-Part of application Ser. No.07/888,564, filed May 22, 1992 (abandoned), which is aContinuation-in-Part application Ser. No. 07/695,506, filed May 6, 1991(abandoned).

TECHNICAL FIELD

The present invention relates generally to new human tumor associatedantigens. This invention is more particularly related to extended type 1chain glycosphingolipids and their uses, e.g., as inmunogens and astumor markers.

BACKGROUND OF THE INVENTION

Despite enormous investments of financial and human resources, cancerremains one of the major causes of death. Current cancer therapies cureonly about 50% of the patients who develop a malignant tumor. In mosthuman malignancies, metastasis is the major cause of death.

Metastasis is the formation of a secondary tumor colony at a distantsite. In most human malignancies, distant metastases are often too smallto be detected at the time the primary tumor is treated. Furthermore,widespread initiation of metastatic colonies usually occurs beforeclinical symptoms of metastatic disease are evident. The size and agevariation in metastases, their dispersed anatomical location, and theirheterogeneous composition are all factors that hinder surgical removaland limit the concentration of anticancer drugs that can be delivered tothe metastatic colonies. Therefore, detection of malignancies prior todissemination of the tumor cells from the primary site is needed toenhance the effectiveness of current cancer therapies.

Aberrant glycosylation has been observed to be a common feature for mostcancer types. Most of the carbohydrate antigens used for the diagnosisof human cancers carry polylactosamine stnictures, i.e., they containGalβ1→3/4GlcNAc. Polylactosamines are usuallv classified into twocategories according to their polylactosamine unit structure. Thepolylactosamine having the Galβ1→3GlcNAc structure is called the type 1chain, and that having the Galβ1→4GlcNAc structure is referred to as thetype 2 chain. The most common tumor-associated antigens found in majorhuman cancers have the lacto-series type 2 chain structure, whichusually has been sialylated and/or fucosylated. Type 1 chain antigensare abundant in normal cells and tissues, and also arecancer-associated. For example, 2-3 sialylated Le^(a) antigen (the CA19-9 antigen defined by the N19-9 antibody) is a cancer-associated type1 chain antigen. However, cancer diagnostic methods based on thedetection of these known antigens have been hampered by high falsepositive and/or high false negative incidences.

Due to the dffficulties in the current approaches to the diagnosis ofcancer, there is a need in the art for improved compositions andmethods. The present invention fills this need, and further providesother related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides isolated compounds andmethods of screening for cancers by detecting such compounds. In oneaspect, the present invention provides an isolated compound, with orwithout fucosyl and/or sialyl residues, having the formula:

    Galβ1→3GlcNAcβ1→3Galβ1→3GlcNAcβ1.fwdarw.(3Galβ1→3GlcNAcβ1→).sub.n 3Galβ1→4Glcβ1→1Cer

wherein n is 0 or an integer of 1 or more, there are at least twofucosyl and/or one or more sialyl residues, Gal represents galactose,Glc represents glucose, GlcNAc represents N-acetylglucosamine, Cerrepresents a ceramide, and wherein said at least two fucosyl residuesare linked to the GlcNAc residues via an α1→4 linkage and/or to theterminal Gal residue via an α1→2 linkage and said one or more sialylresidues are linked to the terminal Gal residue via an α2→3 linkageand/or to one or more of the subterminal GlcNAc residues via an α2→6linkage.

In a further aspect, the present invention provides the above-describedisolated compound having the formula: ##STR1## wherein Fuc representsfucose and NeuAc represents N-acetylneuraminic acid.

In another aspect the invention provides the first-described compoundhaving the formula: ##STR2## wherein Fuc represents fucose and NeuAcrepresents N-acetylneuraminic acid.

In another aspect the invention provides the first described compoundhaving the formula: ##STR3## wherein Fuc represents fucose and NeuAcrepresents N-acetylneuraminic acid.

In one embodiment, the present invention provides an isolated compoundhaving the formula: ##STR4##

In another embodiment, the present invention provides an isolatedcompound having the formula: ##STR5##

In an even further embodiment, the present inv-ention provides anisolated compound having the formula: ##STR6##

Within a related aspect, the present invention provides an isolatedcompound comprising an epitope having the formula: ##STR7##

Within another related aspect, the present invention provides anisolated compound comprising an epitope having the formula: ##STR8##

Within an even further aspect, the present invention provides anisolated compound comprising an epitope having the formula: ##STR9##

In yet other aspects, any of the compounds of the present invention maybe used as an immunogen for the production of polyclonal or monoclonalantibodies.

In another aspect of the present invention, methods for screening forcancer are provided. The methods comprise the steps of: (a) isolating abiological sample from a warm-blooded animal; and (b) testing the samplefor the presence or amount of a compound according to formulae I, II,III, IV, V or VI.

Within a related aspect, the present invention provides the cell lineIMH2, as designated by ATCC No. HB 11026, and the monoclonal antibodyproduced by the cell line (MAb IMH2).

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of HPFTLC immunostaining of upper neutralglycoilpids with MAb NCC-ST-421. Panels A and B, neutral glycolipidsfrom various tumors. Lanes 1-20 and 26-28, glycolipids were obtainedfrom liver adenocarcinoma originating from the primary lesion indicated.Lanes 1-5 and 8-11, colon; lanes 6, 7, 12-14, 26-28, lung; lane 15,breast; lane 16, Hodgkin's disease; lanes 17-19, gall bladder; lane 20,prostate; lane 21, renal cell carcinoma; lane 22, leiomyosarcoma; lane23, embryonal rhabdomyosarcoina; lane 24, breast; lane 25, colon. PanelC, neutral glycolipids obtained from normal tissues. Lanes 1, 6, and 9,liver; lane 2, small intestine; lane 3, spleen; lanes 4-5, kidney; lane7, pancreas; lane 8, placenta; lane 10, lung.

FIG. 2 shows the results of TLC immunostaining of dimeric Le^(a) aftersuccessive enzymatic degradation. Panel A TLC immrunostaining patternwith anti-Le^(a) MAb; Panel B, TLC immunostaining with MAb MNH-1; PanelC, TLC immunostaining with MAb 1B2. Lane 1, type "O" erythrocyte upperneutral fraction; lane 2, Colo205 upper neutral glycolipid fraction;lane 3, dimeric Le^(a) ; lane 4, slow-migrating band "b" afterα-fucosidase treatment of dimeric Le^(a) ; lane 5, fast-migrating band"a" after continued α-fucosidase treatment of dimeric Le^(a) ; lane 6,product formed after β-N-acetylhexosaminidase treatment of lane 5compound; lane 7, product formed after β-galactosidase treatment of lane6 compound.

FIG. 3 depicts a resolution-enhanced 1-D ¹ H-NMR spectrum of dimericLe^(a) (downfield region). Arabic numerals refer to ring protons ofresidues designated by roman numerals in the corresponding structureshown at top of figure. R refers to protons of the sphingosine backboneonly; Cis refers to vinyl protons of unsaturated fatty acids. FucH-5/CH₃ connectivities were confirmed by decoupling.

FIG. 4 depicts chemical ionizationa mass chromatograms of partiallymethylated alditol or hexosaminitol acetates yielded from permethylatedColo205 glycolipid antigen. Separation was performed on a DB-5 bondedphase fused silica column. Peaks identified were (1)2,3,4-tri-O-Me-Fuc-, (2) 2,3,4,6-tetra-O-Me-Gal-, (3)2,3,6-tri-O-Me-Glc-, (4) 2,4,6-tri-O-Me-Gal-, (5) 6-mono-O-Me-GlcNAcMe.Chromatograms are summations of all relevant MH⁺, (MH-32)⁺, and (MH-60)⁺ions.

FIG. 5 depicts a positive ion fast atom bombardment mass spectrum ofpermethylated dimeric Le^(a). The figure is a composite of threeacquisitions optimized for sensitivity under different conditions.Segment from 100-1800 a.m.u. was acquired with NBA only as matrix. Lowersegment from 1800-2500 a.m.u. was acquired with addition of 15-Crown-5to matrix. Insert segment (1900-2500 a.m.u.) was acquired with additionof sodium acetate to matrix. All assignments are nominal monoisotopicmasses.

FIG. 6 depicts a proposed scheme for fragmentation of permethylateddimeric Le^(a). All fragments are assigned nominal, monoisotopic masses.Pseudomolecular ions and additional fragments are listed in Table II.

FIG. 7 graphically illustrates the reactivity of dimeric Le^(a), Le^(a)-Le^(x) and related glycolipids with MAb NCC-ST-421. Solid-phaseradioimmunoassay with MAb ST-421 using serially-diluted dimeric Le^(a),Le^(a) /Le^(x), and various structurally-related glycoilpid antigens.Initial concentration of glycolipids was 100 ng. ST421 was used at aconcentration of 10 μg/ml. The reactivities of twelve glycolipids (A toL) are shown in this figure, corresponding to various structures shownin Table III (below). A, IV³ Galβ1→3[Fuc1→4]GlcNAcIII³ FucnLc₄ (Le^(a)/Le^(x) ; structure 10 in Table III); B, V⁴ III⁴ Fuc₂ Lc₆ (dimericLe^(a) ; structure 11); C, IV³ Galβ1→3[Fucα1→4]GlcNAcnLc₄ (structure 8);D, III⁴ FucLc₄ (structure 1); E, IV³ (Galβ1→3GlcNAcIII³ FucnLc₄(structure 9); F, V³ III³ Fuc₂ nLc₆ (structure 7); G, IV² FucLc₄(structure 2); H, IV³ Galβ1→3GlcNAcnLc₄ (structure 4); I, IV² III⁴ Fuc₂Lc₄ (structure 3); J, nLc₆ (structure 5); K, VI² FucnLc₆ (type 2 H withnLc₆ core; not shown in Table III); L, VI² V³ Fuc₂ nLc₆ (Le^(y) withnLc₆ core; not shown in Table III).

FIG. 8 graphically illustrates the reactivity of MAb IMH2 with variousglycosphingolipids (GSLs). Serial double dilutions of various GSLantigens were added to 96-well flat-bottom assay plates (Probind plate,Falcon) in ethanol and dried. Initial concentration of GSL added to thefirst well was 100 ng/well. MAb binding assay was performed by ELISA asdescribed below. Abscissa, reciprocal of antigen dilution. Ordinate,optical density reading at 490 nm. Paragloboside is abbreviated as "PG".Panel A: reactivity of type 1 chain GSLS. , Le^(b) /Le^(a). ∘, Le^(b)hexasaccharide (IV² FucIII⁴ FucLc₄). Δ, Le^(a) /Le^(a). □ (all of thefollowing showed similar reactivity), type 1 chain PG (Lc₄); H₁ type 1chain (IV² FucLc₄); Le^(a) /Le^(x) (IV³ Galβ1→3[Fuca1→4]GlcNAcβIII³FucnLc₄). Panel B: reactivity of type 2 chain GSLs. , Le^(y) /Le^(x)(VI² FucV³ FucIII³ FucnLc₆). ∘, Le^(y) hexasaccharide ceramide (IV²FucIII³ FucnLc₄). Δ, Le^(x) /Le^(x) (V³ FucIII³ FucnLc₆). □ (all of thefollowing had similar reactivity), H₁ type 2 chain (IV² nLc₄); H₂ type 2chain (IV² FucnLc₆); PG (nLc₄); Le^(x) (III³ FucnLc₄). When GSL antigenswere mixed with cholesterol and phosphatidylcholine in a molar ratio of1:5:3 in ethanol and analyzed as above, the relative degrees ofreactivity were essentially the same as shown in the figure.

FIG. 9 graphically illustrates the MAb-dependent cytotoxic effect onColo205 cells by MAbs IMH2 and ST-421. Panel A: cytotoxic effects atvarious E:T (effector:target cell) ratios. MAbs IMH2 and ST421 werepurified and applied at a concentration of about 30 μg/ml. ⁵¹ Cr-labeledColo205 cells were incubated with different ratios of human peripheralblood leukocytes (HPBL) as effectors. At higher E:T ratios, lysis wasmore conspicuous for both IMH2 and ST-421. ∘, ST-421. , IMH2. ▴,control mouse IgG and D11G10 (IgG₃ anti-Gg3) as non-specific MAbs. PanelB: dependent of cytotoxic effect on MAb concentration at constant E:Tratio 100:1. Maximal cytotoxic effect was observed at a MAbconcentration of 35-70 μg/ml. Control MAb D11G10 (∘) showed no cytotoxiceffect. Panel C: cytotoxic effect with mouse splenocytes as effectorcells. Experimental conditions as in Panel A. ⁵¹ Cr-labeled Colo205cells were incubated with various ratios of mouse splenocytes aseffector cells. ∘, ST-421. , IMH2. Δ, control MAb (D11G10).

FIG. 10 graphically illustrates the CDC (complement-dependentcytotoxicity) effect on Colo205 cells by MAb IMH2. Fresh human serum wasused as complement source. Panel A: ⁵¹ Cr-labeled Colo205 cells wereincubated with about 30 μg/ml of purified IMH2 or ST-421 and variousconcentrations of complement (see abscissa). ∘, ST-421. , IMH2. ▴,control mouse IgG₃ with complement. Panel B: ⁵¹ Cr-labeled Colo205 cellswere incubated with different concentrations of IMH2 (see abscissa) inthe presence of 1:4 diluted human serum as a complement source. , IMI2.∘, control mouse IgG₃ with complement.

FIG. 11 graphically illustrates the inhibitory effect of MAb IMH2 onColo205 cell growth in nude mice. Colo205 cells (1×10⁷) weresubcutaneously injected into the backs of 6-week-old athymic Balb/cmice, followed immediately by injection of 200 μl (≈200 μg) of purifiedIMH2 (1.1 mg/ml) per day for 14 days (shaded bar) (). Other mice weretreated similarly with MAb ST421 (Δ). Control groups were injected withPBS containing similar quantities of non-specific mouse IgG (∘).

FIG. 12 pictorially depicts immunohistological patterns of various humancarcinoma tissues stained by MAb IMH2. A colonic carcinoma, x100. B,colonic carcinoma, x160. C, lung adenocarcinoma, x100. D, livermetastasis from colonic adenocarcinoma. E, F, endometrial carcinoma,x100. It is noted that in each panel adjacent normal tissues were notstained.

FIG. 13 is the ¹ H-NMR spectrum of extended sialyl Le^(a) from chemicalshift at 4.20 ppm to 5.60 ppm covering sugar I(Glc), II(Gal),III(GlcNAc), IV(Gal), V(GlcNAc) and VI(Gal as well as fucose linked toIII GlcNAc identified as F_(III) and fucose linked to V GlcNAc asindicated by F_(IV). In this spectrum, all anomeric proton spectrums ofF_(V) and F_(III) are indicated as F_(V) -1 and F_(III) -1. In addition,spectrum C5 proton of fucoses are indicated by multiple coupling asindicated by F_(III) -5 and F_(V) -5. Spectrum marked as Cis is a Cisdouble bond of sphingosine and R-5 and R-4 indicate spectrum ofsphingosine.

FIG. 14 is a typical ¹ H-NMR spectrum for thiree equatorial protons ofsialic acid of extended sialyl Le^(a).

FIG. 15 is the ¹ H-NMR spectrum of extended sizilyl Le^(a) and shows twomajor spectrums for the N-acetyl group of sialic acid marked as A-NAc.In addition, this figure also shows the spectrum of the N-acetyl groupof GlcNAc marked as GlcNAc-NAc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed towards compounds andmethods relating to the detection of cancers. More specifically, thedisclosure of the present invention shows that lacto-series type 1 chainoccurs in extended forms in cancer tissues.

As noted above, type 1 chain lactosamine (Galβ1→3GlcNAc) is known to beabundant in normal cells and tissues. Although polylactosamine antigenshaving an extended type 2 chain (i.e., Galβ1→4GlcNAc core structure isrepeated) have been detected, those with an extended type 1 chain havenot been detected. Thus, lacto-series type 1 chain has traditionallybeen considered not to occur in extended form.

As disclosed within the present invention, extended forms oflacto-series type 1 chain (i.e., Galβ1→3GlcNAcβ1→[3Galβ1→3GlcNAcβ1→]_(n)3Galβ1→4GalβR, with or without sialyl and/or fucosyl residues) arepresent in cancer tissues. Two representative extended forms oflacto-series type 1 chain were isolated by subjecting a glycolipidfraction (extracted from tumor cells) to preparative column and thinlayer chromatography. Structural determination (by enzymaticdegradation, ⁺ FAB-MS, methylation analysis and ¹ H-NMR spectroscopy)resulted in the identification of the glycosphingolipi(Is (GSLs),dimeric Le^(a) (i.e., Le^(a) -Le^(a)), Le^(b) -Le^(a) and extendedsialyl Le^(a) (sLe^(a) -Le^(a)). The GSL dimeric Le^(a) has thestructure: ##STR10## The GSL Le^(b) -Le^(a) has the structure: ##STR11##Ceramides (Cer) are sphingolipid bases which are acylated on the aminewith a fatty acid.

A slow-migrating sialyl-Lewisa (sLe^(a)) active glycosphingolipid (GSL)was purified to homogeneity from the monosialyl ganglioside fraction ofthe colonic adenocarcinoma cell line Colo205. This compound was purifiedby HPLC and preparative HPTLC in two different solvent systems andstained strongly by TLC immunostaining using the α-sLe^(a) monoclonalantibody (Mab) NKH-1. Mild acid hydrolysis (1% acetic acid, 100° C. for1 hour) yielded a faster migrating component that co-migrated with adimeric-Le^(a) standard GSL and stained strongly by the α-dimeric Le^(a)Mab ST-421. The structures was confirmed by ¹ H-NMR spectroscopy assialyl-dimeric Le^(a) (see structure below). This structure represents anovel tumor-associated GSL and a potential tumor marker. ##STR12##

In addition to the particular glycolipids depicted above, the Le^(a)-Le^(a) Le^(b) -Le^(a) is Le^(a) -Le^(a) epitopes may be present asextended type 1 chains with additional [3Galβ1→3GlcNAcβ1→]_(n) units.Furthermore, the Le^(a) -Le^(a) and Le^(b) -Le^(a) epitopes may becarried by glycoproteins, e.g., high molecular weight mucin-like seraglycoproteins.

Given the teachings provided herein, it would be evident to those ofordinary skill in the art that other extended forms of lacto-series type1 chain compounds may be isolated from biological starting materials,such as cancer tissue, or synthesized chemically (and/or enzymatically)following structural identification. Briefly, the structure ofcarbohydrates bound to either lipids or proteins may be determined basedon degradation, mass spectrometry, including electron-impactdirect-probe (EI) and fast atom bombardment (FAB), and methylationanalysis (techniques described below and, for example, in Nudelman etal., J. Biol. Chem. 261:5487-5495, 1986). Degradation analysis may beaccomplished chemically and/or enzymatically, e.g., by glycosidases. Thecarbohydrate sequence suggested by degradation analysis may bedetermined by methylation analysis (e.g., Hakomori, J. Biochem.55:205-208, 1964) followed by chemical ionization mass spectrometry ofpermethylated sugars (e.g., Stellner et al., Arch. Biochem. Biophys.155:464-472, 1974; Levery et al., Meth. Enzymol. 138:13-25, 1987).Alternatively, or in conjunction with these techniques, EI massspectrometry may be performed on permethylated glycans or after theappropriate degradation of intact glycans (e.g., Kannagi et al., J.Biol. Chem. 259:8444-8451, 1984; Nudelman et al., J. Biol. Chem.263:13942-13951, 1988). Homogeneity of the carbohydrate sequence may bedemonstrated based on various chemical and physical criteria, includingproton NMR spectroscopy of intact or methylated glycan and FAB massspectrometry. Once a carbohydrate structure has been determined, thecarbohydrate or derivatives thereof or non-carbohydrate functionalequivalents thereof may be synthesized using techniques well known tothose of ordinary skill in the art.

The compounds of the present invention may be used as immunogens for theproduction of polyclonal and monoclonal antibodies (MAbs). Polyclonalantibodies may be produced by standard mnethodologies. For example,briefly, polyclonal antibodies may be produced by immunization of ananimal with a compound of the present invention and subsequentcollection of its sera. It is generally preferred to follow the initialimmunization with one or more boosters prior to sera collection. MAbsmay be generally produced by the method of Kohler and Milstein (Nature256:495-497, 1975; Eur. J. Immunol. 6:511-519, 1976). Briefly, the lymphnodes and/or spleens of an animal immunized with a compound of thepresent invention are fused with mnyeloma cells to form hybrid celllines ("hybridomas" or "clones"). Each hybridoma secretes a single typeof immunoglobulin and, like the myeloma cells, has the potential forindefinite cell division. An alternative to the production of MAbs viahybridomas is the creation of MAb expression libraries usingbacteriophage and bacteria (e.g., Sastry et al., Proc. Natl. Acad. Sci.USA 86:5728, 1989; Huse et al., Science 246:1275, 1989). Selection ofantibodies exhibiting a desired specificity may be performed in avariety of ways well known to those of ordinary skill in the art.

It may be desirable to combine a compound of the present invention witha carrier in order to increase their immunogenicity. Suitable carriersinclude inactivated bacteria, keyhole limpet hemocyanin, thyroglobulin,bovine serum albumin and derivatives thereof. For example, all or aportion of the carbohydrate residues of the GSLs Le^(a) -Le^(a) orLe^(b) -Le^(a) may be combined with a carrier. A compound of the presentinvention may be combined with a carrier by a variety of means,including adsorption and covalent attachment.

A representative example of the use of a compound of the presentinvention as an immunogen is the immunization of mice with Le^(b)/Le^(a) antigen. In brief, Le^(b) /Le^(a) isolated from Colo205 cellswas combined with a suspension of acid-treated Salmonella minnesotae,injected via tail vein into Balb/c mice, and the injection repeatedthree times with 10-day intervals. Following the final injection,splenocytes of immunized mice were harvested and fused with myelomacells. A hybridoma, IMH2, which showed preferential reactivity with theimmunogen, was established and deposited with ATCC (American TypeCulture Collection, 12301 Parklawn Dr., Rockville, Md. 20852 USA) asATCC No. HB 11026. The hybridoma produces a MAb IMH2 with an IgG₃isotype.

MAb IMH2 reacts not only with the inununogen used, but also with Le^(y)/Le^(x) antigen, and to a lesser degree with short-chain Le^(y) orLe^(b) with hexasaccharide ceramide (i.e., IV² FucIII³ FucnLc₄ Cer orIV² FucIII⁴ FucLc₄ Cer). It showed high incidence of staning and strongreactivity with carcinomas of colon, rectum, liver, pancreas, andendometriu but no reactivity with normal colonic mucosa at various loci,and minimal reactivity with normal liver, pancreas, or uterineendometrium. Its expression in colorectal tumors and normal cecal tissuewas independent of secretor status, whereas that in normal urotheliumwas dependent on secretor status. MAb IMH2 displayed stronglymphocyte-activated or complement-dependent killing of human coloniccancer Colo205 cells in vitro, and inhibition of Colo205 growth in vivo.Therefore, as disclosed within the present invention, a new extendedtype 1 chain structure, Le^(b) /Le^(a), is a useful tumor markerassociated with carcinomas of colon, rectum, pancreas, liver, andendometrium, and MAb IMH2 has diagnostic and therapeutic applicabilityfor these carcinomas.

Methods for the detection of extended forms of type 1 chain antigen,such as Le^(a) -Le^(a) and/or Le^(b) -Le^(a) antigens, may be used toscreen for cancers. For example, the GSL Le^(b) -Le^(a) and the GSILe^(a) -Le^(a) were detected by TLC immunostaining with MAb IMM and MAbNCC-ST-421 (established according to Watanabe et al., Jpn. J. CancerRes, (Gann) 7:43→52, 1985), respectively, of neutral glycolipidfractions prepared firom various tumor samples. Such samples includetissue from colonic cancer, breast cancer, Hodgkin's disease,gallbladder cancer and embryonal rhabdomyosarcoma The GSL Le^(a)-Le^(a), for example, was not detected in glycolipid fractions fromnormal tissue from spleen, liver, kidney, placenta and lung. Given theteachings provided herein, it would be evident to those of ordinaryskill in the art that a variety of means for detecting tumor-associatedextended type 1 antigens (including the use of binding partners specificfor tumor-associated extended type 1 antigens, such as GSL Le^(a)-Le^(a) and Le^(b) -Le^(a)) could be employed within the methods of thepresent invention. For example, antibodies specific for Le^(a) -Le^(a)or Le^(b) -Le^(a) epitopes may be produced as described above, and thepresence of immunocomplexes may be tested following contact (e.g.,incubation) of such antibodies with a biological sample under conditionsand for a time sufficient to permit the formation of immunocomplexes.

Detection of the presence of immunocomplexes formed between an antigendescribed above and an antibody specific for the antigen may beaccomplished by a variety of known techniques, such as radioimmunoassays(RIA) and enzyme-linked immunosorbent assays (ELISA). Suitableimmunoassays include the double monoclonal antibody sandwich immunoassaytechnique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh, 1970); the "western blot" method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem. 255:4980-4983, 1980); enzyne-linked immunosorbent assays asdescribed by, for example, Raines and Ross (J. Biol. Chem,257:5154-5160, 1982); immunocytochernical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol. 39: 477, 1980); andneutralization of activity (Bowen-Pope et al., Proc. Natl. Acad. Sci.USA 81:2396-2400, 1984). In addition to the immunoassays describedabove, a number of other inununoassays are available, including thosedescribed in U.S. Pat. Nos.: 3,817,827; 3,850,752; 3,901,654; 3,935,074;3,984,533; 3,996,345; 4,034,074; and 4,098,876.

For detection purposes, the antibodies may either be labeled orunlabeled. When unlabeled, the antibodies find use in agglutinationassays. In addition, unlabeled antibodies can be used in combinationwith labeled molecules that are reactive with immrunocomplexes, or incombination with labeled antibodies (second antibodies) that arereactive with the antibody directed against the compound, such asantibodies specific for immunoglobulin. Alternatively, the antibodiescan be directly labeled. Where they are labeled, the reporter group caninclude radioisotopes, fluorophores, enzymes, luminescers, or dyeparticles. These and other labels are well known in the art and aredescribed, for example, in the following U.S. Pat. Nos.: 3,766,162;3,791,932; 3,817,837; 3,996,345; and 4,233,402.

In one preferred embodiment for detecting immunocomplexes, a reportergroup is bound to the antibody. The step of detecting immunocomplexesinvolves removing substantially any unbound antibody and then detectingthe presence of the reporter group. Unbound antibody is antibody whichhas not bound to the antigen.

In another preferred embodiment, a reporter group is bound to a secondantibody capable of binding to the antibodies specific for the antigen.The step of detecting immunocomplexes involves (a) removingsubstantially any unbound antibody (i.e., antibody not bound to theantigen), (b) adding the second antibody, (c) removing substantially anyunbound second antibody and then (d) detecting the presence of thereporter group. For example, where the antibody specific for the antigenis derived from a mouse, the second antibody is an anti-murine antibody.

In a third preferred embodiment for detecting immunocomplexes, areporter group is bound to a molecule capable of binding to theimmunocomplexes. The step of detecting involv(s (a) adding the molecule,(b) removing substantially any unbound molecule, and then (c) detectingthe presence of the reporter group. An example of a molecule capable ofbinding to the immunocomplexes is protein A.

An alternative to the use of labeled antibodies, labeled secondantibodies or labeled molecules reactive with immunuocomplexesgenerally, is an immunoassay employing a labeled antigen. In such anassay ("indirect" or "competitive"), an antigen present in a sample willcompete with labeled antigen for the antibodies.

It will be evident to those of ordinary skill in the art that a varietyof methods for detecting immunocomplexes may be employed within thepresent invention. Reporter groups suitable for use in any of themethods include radioisotopes, fluorosphores, enzymes, luminescers, anddye particles. Further, it will be appreciated that binding partners(other than antibodies) specific for tumor-associated extended type 1antigens of the present invention may be used to test for such antigensand that complexes formed between such binding partners and antigens maybe detected by techniques analogous to those described above forimnmunocomplexes.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 HPTLC Immunostaining and Immunoassay with MA_(B)NCC-ST-421 of Neutral Glycolipids Prepared from Tumors and NormalTissues

A. Monoclonal Antibodies and Immunoassays

MAb ST-421 was established as previously described (Watanabe et al.,Jpn. J. Cancer Res. (Gann) 76:43-52, 1985). MAb MNH-1, which definestype 1 chain N-acetyllactosamine (Galβ1→3GlcNAcβ1→R), was prepared inlaboratory of the inventors; MAb 1B2, which defines type 2 chainN-acetyllactosamine (Galβ1→4GlcNAcβ1→R), was established as previouslydescribed (Young et al., J. Biol. Chem. 256:10967-10972, 1981).Anti-Le^(a) MAb was obtained from Chembiomed Ltd. (Edmonton, Alberta,Canada). Anti-Le^(y) MAb AH6 was established as previously described(Abe et al., J. Biol. Chem. 258:11793-11797, 1983), and did not show anycross-reactivity with Le^(b). Anti-Le^(b) MAb was purchased fromChembiomed Ltd. (Edmonton, Alberta, Canada), and showed cross-reactivitywith type 1 chain H. Another anti-Le^(b) MAb was purchased from Monocarb(Lund, Sweden), and showed reactivity with Le^(b), type 1 chain H, andLe^(y). HPTLC immunostaining was performed using Whatman HPTLC plates(HP-KF) by a modified version (Kannagi et al., J. Biol. Chem.257:4438-4442, 1982; Kannagi et al., J. Biol. Chem. 257:14865-14874,1982) of the method originally described by Magnani et al. (Magnani etal., Anal. Biochem. 109:399-402, 1980).

B. Glycolipid Preparation

All glycolipid samples used were either isolated or synthesizedenzmatically. VI³ NeuAcnLc₆, IV³ NeuAcIII⁴ FucLc₄, VI² FucnLc₆, and IV²FucLc₄ were isolated from human placenta, liver adenocarcinoma, humantype O erythrocytes, and porcine intestine, respectively, afterextraction with IHW (55:25:20) followed by Folch partition,DEAE-Sephadex chromatography, and HPBLC on an latrobeads 6RS-8010 column(Magnani et al., J. Biol. Chem. 257:14365-14369, 1982; Watanabe et al.,J. Biol. Chem, 2548223-8229, 1979; Hakomori et al., J. Immunol.98:31-38, 1967; Stellner et al., Biochemistry 12:656-661, 1973). nLc₆and III⁴ FucLc₄ were prepared by desialylation of VI³ NeuAcnLc₆ and IV³NeuAcIII⁴ FucLc₄, respectively, by heating the samples at 100° C. for 1hr in 1% acetic acid. IV³ GlcNAcnLc₄, IV₃ Galβ1→3-GlcNAcnLc₄, IV₃Galβ1→3[Fuc1→4]GlcNAcnLc₄ and IV³ Galβ1→3[Fuc1→4]GlcNAcIII³ FucnLc₄(Le^(a) -Le^(x)) were prepared by enzyatic synthesis. IV³Galβ1→3GlcNAcIII³ FucnLc₄ was prepared by α-fucosidase treatment of IV³Galβ1→3[Fuc1→4]GlcNAcIII³ FucnLc₄ ; i.e., 100 μg of the glycolipid wasincubated with 0.2 M citrate buffer (pH 4.5) containing 0.05 unitsbovine kidney α-L-facosidase (Sigma Chemical Co., St. Louis, Mo.) for 2hr at 37° C. IV² III⁴ Fuc₂ Lc₄, V³ III³ Fuc₂ nLc₆, and VI² V³ Fuc₂ nLc₆were prepared biosynthetically by α1→3 fucosylation of IV² FucLc₄, nLc₆,and VI² FucnLc₆ (respectively) as substrates, using α1→3/4fucosyltransferase from Colo205. α1→3/4 fucosyltransferase wassolubilized from Colo205 cells by homogenization in two volumes of 50 mMHepes buffer (pH 7.0), 0.5 M sucrose, 1 mM EDTA, and 1% Triton CF-54 ina Potter-Elvehjem homogenizer at 4° C. The homogenate was centrifuged at100,000×g for 1 hr, and the supernatiuat was concentrated to theoriginal volume of cells by dialysis. The enzyme preparation was storedat -80° C. until needed.

Enzymatic α1→3/4 fucosylation was performed in a reaction mixturecontaining 1 mg glycosphingolipid (GSL) substrate, 1 mgdeoxytaurocholate, 10 μmol MnCl₂, 25 μmol Hepes buffer (pH 7.0), 5 μmolCDP-choline, 6,μmol GDP-fucose, and 500 μl enzyme preparation in a totalvolume of 1 ml. The reaction mixture was incubated at 37° C. for 16 hr,then lyophilized, extracted with isopropanol-hexane-water (IHW)(55:25:20) by sonication, and centrifuged. The supernate was subjectedto HPLC on an latrobeads 6RS-8010 column using gradient elution of IHWfrom 55:40:5 to 55:25:20 over 200 min. Two ml fractions were collectedand tubes containing the final product were pooled according to HPTLCmigration in chloroform-methanol-water 50:40:10. GSL bands werevisualized by orcinol spray reagent.

Each GSL with defined structure was characterized by reactivity withspecific MAb(s), i.e., Le^(b) /Le^(a) antigen reacted with anti-Le^(b)MAbs but not with anti-Le^(y) MAb AH6; Le^(y) /Le^(x) reacted with AH6but not with anti-Le^(b) nor anti-Le^(x) MAbs; Le^(a) /Le^(a) and Le^(a)/Le^(x) reacted with anti-Le^(a) MAb as well as with MAb ST-421.

C. TLC Immnunostaining

TLC immunostaining of neutral glycolipid fractions prepared from varioustumor samples showed the presence of a positive band migrating slowerthan Le^(a) -active ceramide pentasaccharide, and cross-reacting withanti-Le^(a) MAb. This band was strongly stained by MAb NCC-ST-421, andwas seen in the majority of tumors so far examined. Examples fromcolonic cancer, breast cancer, Hodgkin's disease, gallbladder cancer,and embryonal rhabdomyosarcoma are shown in FIGS. 1A and 1B. Incontrast, no ST-421-positive band was observed in glycolipid fractionsprepared from normal tissues such as spleen, liver, kidney, placenta, orlung, with the exception of a positive band from extracts of normalsmall intestine and pancreas (FIG. 1C). Thus, presence of thisslow-migrating, Le^(a) -cross-reactive band is highly limited in normaltissues as compared to cancer tissues. In addition, lanes 3, 5, 10, 17and 18 (FIGS. 1A and 1B) show a positive band migrating just above themajor slow-migrating band. This band may represent an extended Le^(a)antigen (e.g., Le^(a) antigen with internal type 1 chain withoutfucosylation).

D. Reactivity of ST-421 with Dimeric Le^(a) and Various RelatedGlycolipids

The solid-phase radioimmunoassay as described by Kannagi et al. (CancerRes. 43:4997-5005, 1983) was used. Solid-phase radioimmunoassay usingST-421 showed the strongest reactivity with both dinieric Le^(a) and theLe^(a) /Le^(x) hybrid glycolipid (FIG. 7). This antibody could notdistinguish between these two antigens, but showed a clear preferencefor them compared to extended Le^(a) or simple Le^(a). Dimeric Le^(x)and various other structurally related GSLs were not reactive with ST421(FIG. 7).

Example 2 Isolation of Dimeric Le^(a) Antigen, Le^(b) -Le^(a) Antigenand Extended Sialyl Le^(a) Antigen

A. preparation of Tumor Tissue

Colo205 cells (ATCC) (Semple et al., Cancer Res 38:1345-1355, 1978) weregrown in RPMI 1640 medium containing 10% fetal calf serum. Cells wereharvested and passed approximately every 7 days. Cells harvested weretrypsinized, centrifuged, washed twice with phosphate-buffered saline(pH 7.4) and counted using a hemocytometer. 4×10⁶ cells mere injectedsubcutaneously into each of 6 athymic (nude) mnice. Tumors(approximately 2 ml each) were excised after 2 weeks and stored frozenat -80° C. until needed.

B. Isolation of the Slow-Migrating. Le^(a) -Active Component (DimericLe^(a)) from Colo205 Tumor

Approximately 200 g of tumors were extracted withisopropanol-hexane-water (IHW) (55:25:20) followed by Folch partition,DEAE-sephadex chromatography and HPLC on an Iatrobeads 6RS-8010 column.Gradient elution of the upper-phase neutral fraction was performed inIHW from 55:40:5 to 55:25:20 over 200 minutes. Two-ml fractions werecollected and pooled according to HPTLC migration inchloroform-methanol-water (50:40:10). The slow- migrating Le^(a) -activefraction (revealed by TLC imunostaining) was further purified bypreparative TLC on Merck HPTLC plates (Silica Gel 60, Merck, Darmstadt,Germany) and used for structural characterization.

C. Isolation of Le^(b) -Le^(a) Antigen

A positive band (by immunostaining with MAb NCC-ST-421 according toExample 1) which migrates just below diferic Le^(a) antigen was purifiedusing the methods described in section B above.

D. Isolation of Extended Sialyl-Le^(a) (or SLe^(a) -Le^(a) )

Examination of monosialo-ganglioside fraction of Colo 205 cells led toisolation or resulted in isolation of one major ganglioside by a highperformance thin layer chromatography technique. The major band wasextracted and characterized. The structure was identified as: ##STR13##This structure was verified by ¹ H-NMR spectroscopy as shown in FIGS.13, 14 and 15.

Example 3 Characterization of Dimeric Le^(a) and Le^(b) -Le^(a) Antigens

A. Enzymatic Degradation

Enzymatic degradation of 1 mg dimeric Le^(a) was performed by sequentialhydrolysis with 0.5 units of α-fucosidase (bovine kidney), 0.5 units ofβ-galactosidase (Cackbean), and 0.5 units of β-N-acetylglucosaminidase(bovine epididymis) (Sigma Chemical Co., St. Louis, Mo.). All reactionswere carried out in 0.2 M sodium citrate (pH 4.5) for 4 hr at 37° C. ina water bath with shaking. Purification of each degradative product wasperformed by preparative HPTLC.

TLC immunostaining of the purified antigen component with various MAbswas performed before and after successive enzymatic degradation.Treatment of the component with bovine kidney a-fucosidase resulted intwo bands: a fast-migrating band "a" (FIG. 2, lane 5), and aslow-migrating band "b" (FIG. 2, lane 4), both of which reacted with MAbMNH-1³ (FIG. 2B) but not with anti-Le^(a) or 1B2 (FIGS. 2A, 2C).Extensive treatment of the original antigen with bovine kidneyα-fucosidase resulted in decreased band b and increased band a. Bands aand b are therefore assumed to be Lc₆ (Table III, structure 6) and III⁴FucLc₆ (structure 13), respectively, based on their reactivity with MAbsand on further enzymatic degradation patterns. Since MNH-1 reactsspecifically with unsubstituted type 1 chain structure(Galβ1→3GlcNAcβ1→3Gal-β1→R), and MAb 1B2 reacts with unsubstituted type2 chain (Galβ1→4GlcNAcβ1→3Gal-β1→R), no type 2 chain stru(cture could bepresent at the terminus. Therefore, treatment of the antigen with bovinekidney α-fucosidase resulted in removal of the fucose linked at thepeniltimate V-GlcNAc residue, which precedes removal of the fucoseresidue linked at the internal (III-GlcNAc) residue.

Jackbean β-galactosidase treatment of the fucosidase-treated product(corresponding to band a) resulted in loss of reactivity with MAb MNH-1(FIGS. 2B, lane 6) and produced a band migrating higher than theα-fucosidase-treated material. This component (i.e., product aftertreatment with α-fucosidase and β-galactosidase) did not react with MAbsMNH-1, 1B2, or ST421, and was further degraded withβ-N-acetylglucosaminidase from bovine epididymis. The product showingTLC migration corresponding to Lc₄ reacted strongly with MAb MNH-1 (FIG.2B, lane 7) but did not react with MAb 1B2 (FIG. 2C, lane 7). Theseresults indicate strongly that not only the terminal but also theinternal carbohydrate core of this glycolipid antigen consists of type 1chain, i.e., extended type 1 structure which is α1→fucosylated at thepenultimate as well as the internal GlcNAc residue. The structure isassumed to be dimeric Le^(a) (Table III, structure 11). This assumptionwas further confirmed by ⁺ FAB-MS, ¹ H-NMR, and methylation analysis asdescribed in sections B, C and D below.

Extended sialyl-Le^(a) on the SLe^(a) -Le^(a) structure was verified byenzymatic degradation with sialidase to yield the same compournd asLe^(a) -Le^(a) as verified by thin layer chromatography as well asimmunostaining with monoclonal antibody ST-421. The original sialylLe^(a) -Le^(a) or extended Le^(a) do not show any reactivity with MAbST-421. However, this compound showed reactivity with MAb directed tosialyl-Le^(a) such as N-19-9, NKH-1 and NKH-2.

B. ¹ H-NMR Spectroscopy

Approximately 1 mg of sample (from Example 2.B.) was deuterium exchangedby repeated lyophilization from DMSO-d₆ /D₂ O (98:2), then dissolved in0.4 ml of this solvent for ¹ H-NMR analysis. One-dimensional spectrawere recorded at 308 and 328±2°K. on a Bruker (Karlsruhe, West Germany)AM-500 Fourier transform spectrometer/Aspect 3000 data system, usingquadrature detection. The sweep width was 5000 Hz, collected over 16Kdata points. The residual HOD resonance was suppressed using apresaturation pulse during the preparatory delay (PD) period. The PD was2.0 sec. A Lorentzian to Gaussian transformation was used for resolutionenhancement.

The downfield portion of the 1-D ¹ H-NMR spectrum of the slow-migratingLe^(a) -active GSL, obtained in DMSO-d₆ /2% D₂ O at 308°K., isreproduced in FIG. 3. Overall, the spectrum was characterized by a highdegree of correlation of resonances with those previously published forLe^(a) -pentaglycosylceramides (Dabrowski et al., Arch. Biochem.Biophys. 210:405-411, 1981) (allowing for temperature differences),particularly for sets of α-Fuc H-1, H-5, and CH₃ resonances, which areknown to be particularly reliable structural reporter groups. It isworth noting that the presence of a small amount (5%-10%) of type 2chain structures was indicated by the α-anomeric resonance at ≈4.85 ppm(≈4.88 ppm at 328°K.), which is diagnostic for H-1 of Fucα↑3 groups inGSLs bearing one or more Le^(x) -haptens as a minor impurity (Levery etal., Carbohydr. Res. 151:311-328, 1986; Levery et al., Carbohydr. Res.178:121-144, 1988; Hakomori et al., J. Biol. Chem. 259:4672-4680, 1984).For the major component, it was found that satisfactory assignments ofglycosyl H-1, and fucosyl H-5 and CH₃, resonances could be made based onthe hypothesis that the GSLs consisted of a repeating type 1 chain(→3Galβ1→3GlcNAcβ1→) unit, with Fucα→4 to GlcNAc groups attached,creating a dimeric Le^(a) structure. The structure and resonanceassignments were made according to the following arguinents.

In the spectrum of this GSL (FIG. 3), a set of downfield saccharideresonances were found at chemical shifts virtually identical to thosefound in the terminal trisaccharide of Le^(a) (Dabrotvski etal., Arch.Biochem. Biophys. 210:405-411, 1981): β-Gal H-1 at 4.321 ppm (³ J₁,2=7.3 Hz); β-GlcNAc H-1 at 4.768 ppm (³ J₁,2 =8.5 Hz); α-Fuc H-1 at 4.781ppm (³ J₁,2 =3.7 Hz). The remaining saccharide H-1 resonances wereconsistent with an internal Le^(a) -pentaglycosylceramide (Dabrowski etal., Arch. Biochem. Biophys. 210:405-411, 1981), provided one assumes adownfield shift of β-Gal IV-1 (δ≈0.05 ppm), and an upfield shift ofIII-1 (δ≈0.05 ppm), on attachment, to O-3 of Gal IV, of the terminalLe^(a) -trisaccharide hapten. The former effect, a glycosylation-induceddownfield shift, is generally observed upon chain elongation, whereasassumption of the latter, a remote shift effect, requires somerationalization based on a knowledge of secondary structure. Two α-FucH-5 resonances, coupled to upfield CH₃ doublets (³ J₅,6 =6.7 Hz), werefound in the spectrum one at a chemical shift virtually identical tothat found in the spectrum of Le^(a) (4.585 ppm), the other shiftedsomewhat downfield (4.636 ppm). Interestinglv, the α-Fuc H-1 resonancesoccur at virtually identical chemical shifts, sinilar to the case withFucα1→3GlcNAc substituents on repeating type 2 chain (Le^(x)) structures(Levery et al., Carbohydr. Res. 151:311-328, 1986; Levery et al.,Carbohydr. Res. 178:121-144, 1988).

According to this analysis, H-5 of the internal Fucα→4 group is in achemical environment similar to that in Le^(a) -pentaglycosylceramide,while that of the outer Fucα1→4 is deshielded. This is in contrast tothe α-Fuc H-1 resonances, which are identical, or to the β-GlcNAc H-1resonances, where that belonging to the innermost saccharide is shiftedupfield relative to its position in Le^(a), while that of the outermostGlcNAc occurs at shifts identical to that in Le^(a). Analogous examplesof long-range "cross-talk" were observed in repeating type 2 Le^(x)-hapten structures (Levery et al., Carbohydr. Res. 151:311-328, 1986;Levery et al., Carbohydr. Res, 18:121-144, 1988), and presumably giveclues to secondary structural interactions between consecutive haptenicunits, although these may be due to the through-space shielding ordeshielding effects of anisotropic groups on saccharides that are notnecessarily in steric contact.

The proposed structural and resonance assignments are summarized in FIG.3 and Table I. The structure was further confirmed by 2-D ¹ H-NMRexperiments, as well as by linkage analysis, by GC-MS, and FAB-MSanalysis of the permethylated GSLs as described below.

C. Methylation Analysis

The remainder of the permethylated sample was hydrolyzed, reduced, andacetylated according to published procedures (Levery and Hakomori, Meth.Enzymol. 138:13-25, 1987). GC-MS analysis of partially methylatedalditol acetates (PMAAs) was perfomed using a 30m DB-5 (0.25 μm i.d.)bonded phase fused silica capillary column as previously described(Clausen et al., J. Biol. Chem. 262:14228-14234, 1987; Ostrander et al.,J. Biol. Chem. 263:18716-18725, 1988; Nudelman et al., J. Biol. Chem.263:13942-13951, 1988).

Linkage analysis was carried out on the putative dimeric Le^(a) GSL bycapillary GC-chemical ionization MS, using the bonded stationary phaseDB-5. PMAAs of 2,3,4-tri-O-Me-Fuc (terminal Fuc); 2,3,4,6-tetra-O-Me-Gal(terminal Gal); 2,3,6-tri-O-Me-Glc (4-linked GLC); 2,4,6-tri-O-Me-Gal(3-linked Gal); and 6-mono-O-Me-GlcNAcMe (3,4-linked GlcNAc) wereclearly identified. These were present in an approximate ratio of2:1:1:2:2 (FIG. 4). A small trace of 3,6-di-O-Me-GlcNAcMe (1%-2% of6-mono-peak), probably arising from unsubstituted type 2 core chaincontaminants, was observed. This analysis was consistent with theproposed structure of dimeric Le^(a), although it would obviously beimpossible to differentiate results for the Le^(a) -hapten from thosefor the isomeric Le^(x) -hapten, since these produce identicalmono-O-Me-GlcNAcMe derivatives. However, the NMR analysis clearly showedthat the Le^(x) -structure is present only as a minor contaminant inthis fraction.

D. ⁺ FAB-MS

A sample of the glycolipid (≈50 μg) was permethylated by the method ofCiucanu and Kerek (Ciucanu and Kerek, Carbohydr. Res. 131:209-217,1984), as modified by Larson et al. (Larson et al., Carbohydr. Res.161:281-290, 1987), except that equal volumes of MeI and DMSO were used(200 μl each). The reaction time was 60 min and MeI was removed byflushing with N₂ for 25 min at 37° C. prior to partitioning betweenCHCl₃ and H₂ O. After washing 3× with H₂ O, the CHCl₃ was dried underN₂, and a portion of the permethylated sample was subjected to ⁺ FAB-MS,performed on a JEOL (Tokyo, Japan) HX-110/DA-5000 mass spectrometer/datasystem. Aliquots of permethylated sample (≈20 μg) in MeOH weretransferred to a FAB target and suspended in 3-nitrobenzyl alcoholmatrix (Meili and Seibl, Org. Mass Spectrom. 19:581-582, 1984; Barber etal., Rapid Commun. Mass Spectrom. 2:18-21, 1988) with and without15-Crown-5 (Holmes and Levery, Arch. Biochem. Biophys. 274:633-647,1989; Isobe et al., Trends Anal. Chem 6:78-81, 1987; Holmes, Arch.Biochem. Biophys. 270:630-646, 1989). Additional experiments wereperformed with addition of sodium acetate to matrix (Egge andPeter-Katalinic, Mass Spectrom. Rev. 6:331-393, 1987; Dell, Adv.Carbohydr. Chem. Biochem. 45:19-72, 1987). Scan range was 100-3000a.m.u.; scan slope 1 min 30 sec; acceleration voltage 10 kV; resolution3000; xenon beam, 6 kV. Three scans were accumulated for each spectrumKI/CsI was used as calibration standard.

Following permethylation, the GSL was analyzed by FAB-MS in the positiveion mode (FIG. 5). The predominant pseudomolecular ions observed (MH⁺,nominal masses of 2374, 2376, and 2406 a.m.u.) were consistent with thecomposition deoxy-Hex₂.Hex₄.HexNAc₂ plus ceramides consisting ofsphingosine/fatty acid combinations dl8:1/h24:1, d18:1/h24:0, andtl8:0/h24:1, respectively. Less abundant pseudomolecular ionscorresponding to other ceramide compositions were also observed (seeTable II). A consistent set of ceramide fragments (m/z 688, 690, 720,etc.) were found in the lower mass end of the spectrum (FIG. 5, TableII). An additional set of less abundant pseudomolecular ions(predominantly at 2200, 2202, and 2232 a-mi.) corresponded to animpurity with the composition deoxyHex.Hex₄.HexNAc₂ in combination withthe same ceramide moieties. The identification of all pseudomolecularion species was confirmed by addition of NaAc to the FAB matrix, whichproduced a mass shift of 22 a.m.u. for each (see inset, FIG. 5, TableII).

A number of sequence-related A₁ -type fragments, produced by cleavage atglycosyl linkages with charge retention of the non-reducing portions(Egge and Peter-Katalinic, Mass Spectrom. Rev. 6:331-393, 1987; Dell,Adv. Carbohydr. Chem. Biochem. 45:19-72, 1987), were observed in highabundance. Key fragments, consistent with the proposed dimeric Le^(a)structure, were found at m/z 638 and 1261, representingdeoxyHex.Hex.HexNAc and deoxyHex₂.Hex₂.HexNAc₂, respectively (see FIG.6). Of particular significance was the secondary fragment at m/z 402,produced by neutral loss of the 3-substituents from N-acetylhexosaminylresidues at the non-reducing ends of both of the major primary fragments(i.e., 638-HexOH and 1261-deoxyHex.Hex.Hex.NAc.HexOH) (Egge andPeter-Kataliic, Mass Spectrom. Rev. 6:331-393, 1987; Dell, Adv.Carbohydr. Chem. Biochem. 45:19-72, 1987). These are consistent with theGalβ1→3GlcNAc linkages of type 1 chain repeating core units. In the ⁺FAB mass spectrum of the isometric repeating type 2 chain dimeric Le^(x)structure, the abundant loss of the 3-liinked Fuc residues as deoxyHexOHproduces major fragments at m/z 432 and 1055, as reported previously(Holmes and Levery, Arch. Biochem. Biophys. 24:633-647, 1989). Anotherkey difference from the mass spectrum of dimeric Le^(x) was the abundantgroup of ceramide-containing fragments at m/z 1375, 1515, 1517, and1547. These must be produced by neutral loss of the 3-linked substituentfrom the internal HexNAc residue of the pseudomolecular ions(MH-deoxyHex.Hex.HexNAc.HexOH). An additional fragment at m/z 1073 isbelieved to result from β-cleavage (Dell, Adv. Carbohydr. Chem. Biochem.45:19-72, 1987) of a 4-linked Fuc residue from the primary fragment atm/z 1261 (1261-189+1).

The group of ceramide-containing fragments observed primarily at m/z2028, 2140, 2170, and 2200 (overlapping with the monofucosyl MH⁺cluster) appeared to result from uniform loss of 206 a.m.u. from thedifucosyl MH⁺ ions. Since this is seemingly consistent with loss ofdeoxyHexOH from the difucosyl MH⁺ ions, it could be taken as anindication of sonle impurity of type 2 chain structure losing 3-linkedFuc as a neutral fragment. However, the relative abundance isinconsistent with the small quantity of type 2 chain Le^(x) observed inthe ¹ H-NMR spectrum. Moreover, these fragments were not observed insuch abundance even from the pseudomolecular ions of pure permethylateddimeric Le^(x) under similar conditions (Holmes and Levery, Arch.Biochem. Biophys. 274:633-647, 1989). Thus, it is unclear at this timewhy they are produced in this spectrum, in seeming contradiction toprevious observations of preferred neutral loss of 3-linked substituentson HexNAc. It may be related to an unusual steric condition peculiar tothe repeating dimeric Le^(a) structure.

Finally, fragments at m/z 1087 and 1291 were consistent with thepresence of some monofucosylated impurity. Similar fragments wereobserved in the spectrum of an isomeric synthetic monofucosyl type 2chain structure (Holmes and Levery, Arch. Biochem. Biophs. 274:633-647,1989). However, the relatively low abundance of ion at m/z 464(Hex.HexNAc) indicates that, in this case, the single deoxyHex residuemust be attached primarily to the subterminal, rather than internal,HexNAc.

E. Le^(b) -Le^(a) Antigen

The structure of the isolated Le^(b) -Le^(a) antigen was confirmed usingthe analytical techniques described in sections A-D above.

                                      TABLE I                                     __________________________________________________________________________    Chemical shifts (ppm from tetramethylsilane) and .sup.3 J.sub.1,2             coupling constants (Hz) of glycosyl H-1 resonances.sup.a                      for the slow-migrating Le.sup.a -active GSL in dimethylsulfoxide-d.sub.6      at 308° K. and 328° K.                                          T (° K.)                                                                    ##STR14##                                                                __________________________________________________________________________    308°                                                                       4.321(7.3)                                                                          4.781(3.7)                                                                          4.768(8.5)                                                                          4.366(7.3)                                                                          4.781(3.7)                                                                          4.722(7.9)                                                                          4.269(6.7)                                                                          4.203(7.3)                      328°                                                                       4.341(7.3)                                                                          4.799(3.7)                                                                          4.800(7.9)                                                                          4.382(7.3)                                                                          4.799(3.7)                                                                          4.770(7.9)                                                                          4.284(7.3)                                                                          4.208(7.9)                      __________________________________________________________________________     .sup.a Additional resonances were found as follows: A) at 308° K.,     α-Fuc F.sub.III -5, 4.585 ppm; F.sub.V -5, 4.636 ppm; F.sub.III -6      (CH.sub.3), 1.001 ppm; F.sub.V -6 (CH.sub.3), 1.006 ppm; β-GlcNAc        NAc, 1,816 and 1.834 ppm; B) for 328° K., α-Fuc F.sub.III -5     4.544 ppm; F.sub.V -5, 4.589 ppm; F.sub.III -6 (CH.sub.3), 1.019 ppm;         F.sub.V -6 (CH.sub.3), 1.024 ppm; β-GlcNAc NAc, 1.824 and 1.841 ppm;     # all α-Fuc = .sup.3 J.sub.5,6 = 6.7 Hz.                           

                                      TABLE II                                    __________________________________________________________________________    Nominal masses (a.m.u.) calculated for major ceramide-containing ions in      the .sup.+ FAB mass spectrum of                                               permethylated dimeric Le.sup.a glycosphingolipid from Colo205 cells.          sphingosine                                                                         fatty acid                                                                         Cer.sup.+                                                                        MH.sup.+                                                                         [MH-R.sub.1.sup.a ].sup.+                                                          [MH-R.sub.2.sup.b ].sup.+                                                           MNa.sup.+                                                                         MH.sup.+  (monofuc).sup.c                                                             MNa.sup.+  (monofuc)                  __________________________________________________________________________    d18:1 16:0 548                                                                              2234                                                                             2028 1375  2256                                                                              2060    2082                                  d18:1 24:1 658                                                                              2344                                                                             2138 1485  2366                                                                              2170    2192                                  d18:1 24:0 660                                                                              2346                                                                             2140 1487  2368                                                                              2172    2194                                  d18:1 h24:1                                                                              688                                                                              2374                                                                             2168 1515  2396                                                                              2200    2222                                  d18:1 h24:0                                                                              690                                                                              2376                                                                             2170 1517  2398                                                                              2202    2224                                  t18:0 h24:1                                                                              720                                                                              2406                                                                             2200 1547  2428                                                                              2232    2254                                  __________________________________________________________________________     .sup.a R.sub.1 = deoxyHexOH                                                   .sup.b R.sub.2 = deoxyHex.Hex.HexNAc.HexOH                                    .sup.c monofuc = monofucosylated impurity                                

                                      TABLE III                                   __________________________________________________________________________    Structures of glycolipids with fucosylated lacto-series type 1 and type 2     chain.                                                                        __________________________________________________________________________      III.sup.4 FucLc.sub.4 (Le.sup.a penta)                                                              ##STR15##                                               IV.sup.2 FucLc.sub.4 (H type 1)                                                                     ##STR16##                                               III.sup.4 IV.sup.2 Fuc.sub.2 Lc.sub.4 (Le.sup.b)                                                    ##STR17##                                               IV.sup.3 Galβ1→3GlcNAcnLc.sub.4                                                        Galβ1→3GlcNAcβ1→3Galβ1                           →4GlcNAcβ1→3Galβ1→4G                           lcβ1β→1Cer                              nLc.sub.6            Galβ1→4GlcNAcβ1→3Galβ1                           →4GlcNAcβ1→3Galβ1→4G                           lcβ1β→1Cer                              Lc.sub.6             Galβ1→3GlcNAcβ1→3Galβ1                           →3GlcNAcβ1→3Galβ1→4G                           lcβ1β→1Cer                              III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6 (dimeric Le.sup.x)                                            ##STR18##                                               IV.sup.3 Galβ1→3[Fucα1→4]GlcNAcnLc.sub.4                                   ##STR19##                                               IV.sup.3 Galβ1→3GlcNAcIII.sup.3 FucnLc.sub.4                                            ##STR20##                                             10.                                                                             IV.sup.3 Galβ1→  3[Fucα→1→4]GlcNAcIII.su      p.3 FucnLc.sub.4 (Le.sup.a /Le.sup.x)                                                               ##STR21##                                               III.sup.4 V.sup.4 Fuc.sub.2 Lc.sub.6 (dimeric Le.sup.a)                                             ##STR22##                                               V.sup.4 FucLc.sub.6                                                                                 ##STR23##                                               III.sup.4 FucLc.sub.6                                                                               ##STR24##                                               Le.sup.y                                                                                            ##STR25##                                               Le.sup.b /Le.sup.a                                                                                  ##STR26##                                               Le.sup.y /Le.sup.x (trifucosyl Le.sup.y)                                                            ##STR27##                                               SLe.sup.a -Le.sup.a (extended sialyl-Le.sup.a)                                                      ##STR28##                                             __________________________________________________________________________

Example 4 MA_(B) IMH2

A. Production

MAb IMH2 was established after immunization of Balb/c mice with Le^(b)/Le^(a) antigen (Table III, structure 15) isolated from Colo205 cells(isolated as described in Example 2 above) and adsorbed on Salmonellaminnesotae (adsorption according to Young et al., J. Exp. Med.150:1008-1019, 1979). Forty μg Le^(b) /Le^(a) was dissolved in 100 μlethanol and mixed with 1.6 ml PBS (pH 7.4). This solution was combinedwith 500 μl of a suspension containing 500 μg acid-treated S. minnesotaeand incubated at 37° C. for 30 min with occasional shaking. Thesuspension was freeze-dried, and the dried residue was re-suspended in 2ml water, divided into 8 vials, and frozen until use. Each vialcontained approximately 5 μg of GSL antigen and 62 μg of bacteria in 250μl PBS. Contents of each vial were injected via tail vein into6-week-old female Balb/c mice on four occasions, with 10-day intervals.Two mice were immunized at a given time. Three days after the lastinjection, splenocytes of immunized mice were harvested and fused withNS/1 myeloma cells according to the procedure originally described byYoung et al. (J. Exp. Med, 150:1008-1019, 1979). Hybridomas wereselected using 96-well plates coated withphosphatidylcholine-cholesterol-Le^(b) /Le^(a) 5:3:1 by weight. Thequantity of Le^(b) /Le^(a) added was approximately 50 ng/welL Reactivitywas determined by ELISA assay. Clones showing preferential reactivitywith the itnmunogen were subcloned repeatedly until a stable clone wasestablished. The isotype of MAb IMH2 was determined to be IgG₃.

B. Imm-unochemical Characterization of MAb IMH2 Epitope

The binding specificity of MAb IMH2 was tested by thin-layerchromatography immunostaining of various GSL antigens by the methodpreviously described by Magnani et al. (Anal. Biochem. 109:399-402,1980), and modified by Kannagi et al. (Cancer Res. 43:4997-5005, 1983).IMH2 reactivity was also determined by antibody binding on solid-phaseantigen as follows. Each GSL antigen, dissolved in ethanol (100 ng/50μl), was placed in a well, serially diluted with ethanol to 0.1 ng/50 μlper well in a flat-bottom 48-well plate (Falcon, Becton-Dickinson,Lincoln Park, N.J.), and dried. The dried antigen in each well was thenincubated with 1% bovine serum albumin in PBS for 1 hr at roomtemperature, followed by reaction with IMH2 (5 μg/ml) for 18 hr at 4° C.Each well was washed with PBS, followed by incubation with biotinylatedsecond antibody and avidin-peroxidase conjugate as prepared in the ELISAassay kit. The orange color developed in each well was read by automated"ELISA reader" (EL312 Biokinetics Reader, Biotek Instruments, Winooski,Vt.), and optical density at 490 mm for each well was recorded.

Studies on IMH2 binding (Table IV) to various GSL structures (Table III)revealed that the MAb reacts strongly with Le^(b) /Le^(a) (structure 15;FIG. 8A) as well as Le^(y) /Le^(x) (structure 16; FIG. 8B). It alsoreacted, but more weakly, with hexasaccharide ceramide having Le^(y)(structure 14) or Le^(b) (structure 3) determinants (FIG. 8). It reactedweakly with Le^(a) /Le^(a) antigen (FIG. 8A), but did not react withLc₄, H type 1 chain (IV² FucLc₄), Le^(a) /Le^(x) (FIG. 8A), H₁ (IV²FucnLc₄), H₂ (VI² FucnLc₆), Le^(x) (III³ FucnLc₄), or nLc₄ (FIG. 8B).IMH2 reacted equally well with Le^(y) /Le^(x) (VI² FucV³ FucIII³FucnLc₆) as with Le^(b) /Le^(a), and reacted weakly with Le^(x) /Le^(x)(V³ FucIII³ FucnLc₆) (FIG. 8B).

                  TABLE IV                                                        ______________________________________                                        Reactivity of MAb IMH-2 with various                                          glycosphingolipid (GSL) antigens.                                             GSL         IHM-2 Reactivity                                                  ______________________________________                                        Le.sup.a /Le.sup.a                                                                        -                                                                 Le.sup.b /Le.sup.a                                                                        ++                                                                Le.sup.y /Le.sup.x                                                                        ++                                                                Le.sup.x /Le.sup.x                                                                        -                                                                 Le.sup.a /Le.sup.x                                                                        -                                                                 Le.sup.y    +                                                                 Le.sup.b    +                                                                 ______________________________________                                    

Anomeric protons for two fucoses F_(V) -1 and F_(III) -1 of extendedLe^(a) (or SLe^(a) -Le^(a) ) showed a sharp spectrum at 4.8 ppm which isquite distinct from type 1 chain anomeric spectrum of fucoses linked totype 2 chains. All other spectrum for anomeric protons of III GlcNAc, IVGlcNAc, V GlcNAc and VI Gal are distinctly different from type 2 chains.These characteristics are in accordance with typical type 1 chains asdescribed above in Example 3, Item B.

C. In vitro Cytotoxicitv of IMH2

1. Cell Lines

Colo205 was originally obtained from American Type Culture Collection(ATCC) and cultured in RPMI-1640 medium supplemented with 10% fetal calfserum, 1 mM L-glutamine, 100 IU/mi penicillin, and 10 μg/mlstreptomycin. Human epidermoid carcinoma A431 cell line (MacLeod et al.,J. Cell. Physiol. 127:175-182, 1986) was originally donated by Dr. CarolMacLeod (Gildred Cancer Facility, UCSD School of Medicine. San Diego,Calif.). This cell line expresses Le^(a), Le^(b), Le^(x), Le^(y), andALe^(b) on the EGF receptor (Gooi et al., Biosci. Reports 5:83-94,1985). A431 cells were cultured in Dulbecco's modified Eagle's medium(Irvine Scientific, Santa Ana, Calif.) supplemented with 5% fetal calfserum, 1 mM glutamine, 110 mg/l sodium pyruvate, 100 IU/ml penicillin,and 10 μg/ml streptomycin. Cells (about 5×10⁵ /ml) were seeded andharvested at confluency by EDTA treatment followed by washing with PBScontaining Ca²⁺ and Mg²⁺. These were used as target cells in in vitrocytotoxdcity assay, or used for testing tumorigenicity in nude mice bysubcutaneous inoculation of 5×10⁶ cells. Human erythroleukemia K562cells (Lozzio et al., Blood 45:321-334, 1975) were used as controls fornatural killer (NK) activity of lymphocytes used in the assay system.

2. Antibody-Dependent Cellular Qtotoxicity (ADCC) andComplement-Dependent Qtotoxicity (CDC)

For the ADCC assay, human peripheral blood leukocytes (HBBL) (used aseffector cells) were obtained from bufly coat fraction of blood fromhealthy volunteer donors. Briefly, mononuclear cells were separated bycentrifugation through Ficoll-Hypaque gradient solution at 2000 rpm for20 min (Mishell et al., in Mishell, B. B and Shiigi, S. M. (eds.),Selected Methods in Cellular Immunology, pp. 3-27, W. H. Freeman & Co.,San Francisco, Calif., 1980). Mouse splenocytes and mouse peritonealmacrophages (effector cells) were prepared as previously described byMishell et al., with some modification as follows. Target cells (5×10⁶)were labeled by incubation with 100 μl of ⁵¹ Cr for 90 min at 37° C.After washing (3×) and incubation (1 hr at 37° C.), cells (1×10⁶ ml)were suspended in RPMI-1640 supplemented with 25 mM HEPES buffer and 3%bovine serum albumin. Twenty μl of labeled cells, 100 μl of IMH2 orST-421, ad 100 μl of effector cell suspension were mixed into MicrotiterU-bottom plates (Corning, N.Y.). Non-specific mouse Ig (Sigma, St.Louis, Mo.) was used as a negative control. After 4 hr incubation, theplates were centrifuged (500×g, 2 min) with a hanging plate-holderassembled in a centrifuge, and radioactivity in 100 μl supernatant ineach well was measured with a gamma counter. Each experimental group wastested in triplicate. Percent specific lysis was calculated according tothe formula ([A-B]×100)/C, where A=cpm in lysed experimental cells;B=cpm in unlysed target cells; C=cpm in total target cells. Spontaneousrelease never exceeded 15% of maximally releasable labeledradioactivity.

For CDC, ⁵¹ Cr-release assay was performed using a procedure similar tothat for ADCC, except that 100 μl of diluted human serum was added as acomplement source instead of effector cells. The serum was inactivatedat 56° C. for 30 min and used as a control. Percent specific lysis wascalculated as described above.

Since Colo205 cells have been characterized as expressing extended type1 chain Le^(a) /Le^(a) and Le^(b) /Le^(a) antigens, which react stronglywith MAbs ST-421 and IMH2, respectively, cytotoxic effect of IMH2against Colo205 was evaluated and compared to that of ST-421. Both MAbsshowed strildng ADCC killing of Colo205 cells. This killing wascorrelated with effector:target cell (E:T) ratio (FIG. 9A) and with MAbconcentration (FIG. 9B). The cytotoxic effect was maximal at an E:Tratio of 100:1-200:1, and at a MAb concentration of 35-70 μg/ml. Controlmouse IgG and other non-specific MAbs showed no cytotoxic effectregardless of E-T ratio or MAb concentration (FIGS. 9A, 9B). When thesame cytotoxicity test was performed with mouse splenocytes, thecorresponding values were only 7% and 17% lysis (E:T ratio 200:1, MAbconcentration 30 μg/ml) (FIG. 9C). The MAbs showed a weak cytotoxiceffect against A431 cells (Table V). Comparison of maximumIMH2-dependent lysis of Colo 205, A431, and K562 cells is shown in TableV. High lysis values (e.g., 65% and 94% lysis of Colo205 cells with IMH2and ST421, respectively) were only pronounced in the presence of HPBL;values were much less with moiise splenocytes, as observed previouslywith ST-421 (Watanabe et al., Cancer Res. 51:2199-2204, 1991). CDCmediated by IMH2 and ST-421 was similarly correlated with complementconcentration (FIG. 10A) and with MAb concentration (FIG. 10B).

                  TABLE V                                                         ______________________________________                                        MAb-dependent cytotoxic effect on Colo205, A431, and K562                     cells by MAbs ST-421 and IMH2.                                                              Percent lysis.sup.a                                                      Antibody/  eff. cell +                                                                             eff. cell +                                                                           eff. cell -                             Target Cell                                                                            Reactivity.sup.b                                                                         MAb +     MAb -   MAb +                                   ______________________________________                                        Colo205  ST-421 +   94.5      2.7     0.8                                              IMH2 +     65.0      2.7     0.7                                     A431     ST-421 ±                                                                              14.4      10.9    1.1                                              IMH2 ±  7.6       9.2     0.6                                     K562.sup.c                                                                             ST-421 -   48.2      36.2    0.5                                              IMH2 -     44.8      36.2    0.3                                     ______________________________________                                         .sup.a Percent lysis at E:T (effector:target cell) ratio of 100:1 with        IMH2 (35 μg/ml) and ST421 (x100 diluted ascites).                          .sup.b Determined by flow cytometry. +, positive; ±, weakly positive;      -, negative.                                                                  .sup.c The high cytotoxic effect of K562 cells is also observed in the        absence of MAb, and is considered to reflect natural killer cell activity

D. In vivo Tumor Suppression

Colo205 and A431 cells used for in vivo experiments were grown in vitro,washed 2× with medium, and reconstituted at the desired cell density inPBS. Cells (5×10⁶ /100 μl) were subcutaneously injected into the backsof 5- to 7-week-old athymic Balb/c mice, and intraperitonealadministration of MAb was started immediately after injection. PurifiedIMH2 (1.1 mg/ml) or ST421 in ascites fluid with correspondingconcentration of IgG (1.1-1.2 ng/ml) at a dosage of 0.2 ml/animal wereintraperitoneally injected 1×/day for 2 weeks. Width and length oftumors were measured by the same observer 3×/week. Tumor weight wasestimated as (width² ×length)/2. Control aninials received ascitesprotein produced by mouse myeloma cell line NS1 in Balb/c mice. Sevenmice per group were used for each experiment, and experiments were runin duplicate. Mean values of tumor weight based on the duplicateexperiments were plotted.

Both MAbs IMH2 and ST-421 showed striking inhibition of Colo205 tumorgrowth in nude mice (FIG. 11). In contrast, both MAbs showed minimalinhibitory effect on A431 tumor growth. Thus, high expression of thedefined antigen appears to be essential for susceptibility toantibody-dependent inhibition of tumor growth in vivo.

E. Reactivity of IMH2 With Various Tumors and Normal Tissues

Various tumors and adjacent normal tissues were obtained from surgicalspecimens fixed with formalin and paraffin-embedded. In addition, normaltissues and some tumor tissues from brain, thymus, lung, liver, stomach,colon, kidney, adrenal gland, spleen, pancreas, uterus (withendometrium), and skin were obtained by fresh necropsy from accidentvictims. Both surgical and necropsy specimens were provided through thecourtesy of the Department of Pathology, Swedish Medical Center,Seattle, Wash., and Ms. Debbie Bennett of The Biomembrane Institute.Samples were sectioned (3 μM thickness), deparaffinized with xylene,dehydrated in ethanol, treated with primary MAb, subsequently treatedwith biotinylated secondary MAb and peroxidase-conjugated avidin, andstained with 3', 3'-diaminobenzidine. Endogenous peroxidase activity wasblocked by treatment of sections with 0.3% H₂ O₂ for 20 min. Somesections were incubated with mouse IgG as a negative control.Biotinylated goat anti-mouse IgM, avidin, and biotin were fromVectastain (Burlingame, Calif.).

MAb IMH reacted strongly and with high incidence with tumors from colon,rectum, liver, pancreas, and endometrium (Table VI). In contrast, itshowed no reactivity with normal mucosae of distal colon and rectum,including crypt regions and goblet cells. It reacted with lungadenocarcinoma, but not with large cell or small cell carcinoma One outof 5 cases of squamous cell carcinoma showed strong positive reactivity.MAb IMH2 did not react with tissues of normal brain, lung, spleen, skin,or with various blood cells including granulocytes.

Observed locations of normal tissues with strong staining were asfollows: Hassall's bodies and epithelial reticular cells of thymus(thymocytes were negative); mucous epithelium and secretory glands ofgastric mucosa (lamina propria, serosa, and muscle layer were negative);both medulla and cortex of adrenal glands. Locations of normal tissueswith moderate to weak positive staining were: epithelial cells ofproximal and distal convolutions of kidney (other parts were negative);cells in Langerhans' islets in pancreas (other parts of pancreas werenegative); cecal mucosa; urothelium. Very weak staining was observed forhepatocytes (other parts of liver, infralobular connective tissue,central vein, bile duct, and Kupffer's cells were negative). Theseresults are summarized in Table VI, and some typical positive stainingpatterns are shown in FIG. 12.

                  TABLE VI                                                        ______________________________________                                        Immunohistological staining by MAb IMH2                                       of normal tissues and carcinomas.                                             Tissue      Staining                                                                              Localization/comments                                     ______________________________________                                        Normal                                                                        brain       -                                                                 lung        -       including broncheolar epithelia                           spleen      -                                                                 rectum      -       including crypt area                                      colon       -       -11/12, ±1/12                                          cecum       +                                                                 skin        -                                                                 granulocytes                                                                              -                                                                 lymphocytes -                                                                 pancreas    +       + in islets of Langerhans; others -                       liver       ±    faintly +/± hepatocytes; others --                     thymus      ++      ++ in Hassal's bodies, epithelial and                                         reticular cells; - in thymocytes                          stomach     +++     mucosa, glandular cells (see text)                        kidney      +       weakly + in tubular epithelia (see text)                  adrenal glands                                                                            +++                                                               uterus/endometrium                                                                        -/+     - or weakly + in endometrium;                                                 -9/15, ± 2/15, + 4/15 (total positive                                      cases 4/15 = 27%)                                         Carcinomas                                                                    colon/rectum                                                                              +/+++   +++4, ++6, +4, ±1, -1 (total                                               positive cases 14/16 = 88%)                               liver (primary)                                                                           ++      2/3                                                       pancreas    +++     2/2                                                       lung adenocarcinoma                                                                       ++      2/4                                                       squamous    +       1/5                                                       large cell  -       0/3                                                       small cell  -       0/5                                                       endometrium -/+++   +++4, ++11, +6/24, ±/-3 (total                                             positive cases 21/24 = 88%)                               ______________________________________                                    

F. Reactivity of IMH2 With Normal and Maliegnant Colonic and BladderTissues From Patients With Known Lewis and Secretor Status

Expression of Le^(b) and Le^(y) determinants is correlated with secretorstatus of the individual (Sakamoto et al., Molec, Immun. 21:1093-1098,1984; .O slashed.rntoft et al., J. Urol 138:171-176, 1987), whereasexpression of Lewis antigens in some tumors is unrelated to host Lewisstatus (.O slashed.rntoft et al., Lab. Invest. 58:576-583, 1988; .Oslashed.rntoft et al., Blood 71:1389-1396, 1991). Therefore, reactivityof MAb IMH2 with normal and malignant colonic and bladder tissues frompatients with known Lewis and secretor status was studied. Results aresummarized in Tables VII and VIII. IMH2 was reactive with rectal tumorsbut not with normal rectal tissue, and this reactivity was unrelated tosecretor status. Conversely, IMH2 was reactive with normal cecum butless so with the single cecal tumor sample studied. These resultssuggest that the trend of IMH2 epitope expression in normal andmalignant colonic tissues is similar to the well-established expressionpattern of ABH antigens. Genuine Lewis-negative (Le^(a-b-)) individuals(.O slashed.rntoft et al., Lab. Invest, 58:576-583, 1988), expressedIMH2 epitope in both normal and malignant colonic tissues (Tables VIIand VIII).

IMH2 epitope is expressed in normal urothelium, but its expression isdiminished to varying degrees in bladder tumors. There seems to be acorrelation with grade of atypia, i.e., IMH2 epitope expression islowest in highly invasive tumors. Again, this trend is similar to thatof ABH antigen expression in normal and malignant bladder tissues.However, in contrast to colonic tissues, IMH2 epitope expression inbladder tissues from blood group A individuals is correlated withsecretor status. Genuine Lewis-negative (Le^(a-b-)) individualsexpressed IMH2 epitope in both normal and malignant bladder tissues.

                  TABLE VII                                                       ______________________________________                                        Immunohistological staining by MAb IMH2 of normal and malignant               colonic tissues: Relationship with host Lewis status.                                    Normal      Malignant                                                         rectum cecum    rectum   cecum                                     ______________________________________                                        A Le.sup.a-b+                                                                              0/5      1/1      3/4    1/1                                     A Le.sup.a+b-                                                                              0/4      ND       2/2    ND                                      O Le.sup.a-b+                                                                              0/2      ND       2/3    ND                                      O Le.sup.a+b-                                                                              0/2      ND       1/1    ND                                      genuine Le.sup.a-b-                                                                        0/1      1/1.sup.a                                                                              1/1    0/1                                     non-genuine Le.sup.a-b-                                                                    0/2      ND       1/1    0/1                                     ______________________________________                                         Figures indicate positive specimens divided by total specimens examined.      ND = not determined. For Le.sup.a-b-  individuals (genuine and                nongenuine), phenotypic status was determined by α1→4            fucosyltransferase activity in saliva, and erythrocyte reactivity with        antiLe.sup.a and -Le.sup.b MAbs. Definitions of phenotypes may be found i     Holmes et al., Arch. Biochem. Biophys.  274:14-25, 1989, and .O               slashed.rntoft et al.,  # Lab. Invest.  58:576-583, 1988.                     .sup.a Nonsecretor.                                                      

                  TABLE VIII                                                      ______________________________________                                        Immunohistological staining by MAb IMH2 of normal and                         malignant bladder tissues: Relationship with host Lewis status.                                 Bladder carcinoma                                                       normal  noninvasive invasive                                      ______________________________________                                        A Le.sup.a-b+ 4/4       1/1         1/2                                       A Le.sup.a+b- 0/2       1/2         1/3                                       O Le.sup.a-b+ 1/1       1/1         1/2                                       O Le.sup.a+b- 2/2       1/1         0/1                                       genuine Le.sup.a-b-                                                                         2/2       ND          0/1                                       non-genuine Le.sup.a-b-                                                                     ND        ND          ND                                        ______________________________________                                         Main footnote as for Table VII.                                          

From the foregoing, it will be evident that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modification may be made without deviating fromthe spirit and scope of the invention.

We claim:
 1. An isolated compound having the formula: ##STR29## whereinR¹, R², R³ and R⁴ are fucosyl residues; R⁵, R⁶, R⁷ and R⁸ are sialylresidues, provided that R¹ and R⁵ are not present together; n is 0 or aninteger of 1 or more; when n is 1, the compound has 1 or more sialylresidues; Gal is galactose, GlcNAc is N-acetylglucosamine, Glc isglucose and Cer is ceramide.
 2. The isolated compound of claim 1,wherein there are at least two sialyl residues.
 3. The isolated compoundof claim 1, having the formula: ##STR30## wherein Fuc represents fucoseand NeuAc represents N-acetylneuraminic acid.
 4. The isolated compoundof claim 1 having the formula: ##STR31## wherein Fuc represents fucoseand NeuAc represents N-acetylneuraminic acid.
 5. The isolated compoundof claim 1 having the formula: ##STR32## wherein Fuc represents fucoseand NeuAc represents N-acetylneuraminic acid.
 6. The isolated compoundof claim 1 having the formula: ##STR33## wherein Fuc represents fucose.7. The isolated compound of claim 1 having the formula: ##STR34##wherein Fuc represents fucose.
 8. The isolated compound of claim 1having the formula: ##STR35## wherein NeuAc representsN-acetylneuraminic acid, and Fuc represents fucose.
 9. A pharmaceuticalcomposition comprising an adjuvant and either(a) an isolated glycolipidor glycoprotein compound comprising an epitope having a formula##STR36## wherein NeuAc represents N-acetylneuraminic acid, Galrepresents galactose, GlcNAc represents N-acetylglucosamine, Glcrepresents glucose, and Fuc represents fucose, or (b) the compound ofany one of claims 1-8.